Bile salt metabolism is not the only factor contributing to Clostridioides (Clostridium) difficile disease severity in the murine model of disease

ABSTRACT

Susceptibility of patients to antibiotic-associated C. difficile disease is intimately associated with specific changes to gut microbiome composition. In particular, loss of microbes that modify bile salt acids (BSA) play a central role; primary bile acids stimulate spore germination whilst secondary bile acids limit C. difficile vegetative growth. To determine the relative contribution of bile salt (BS) metabolism on C. difficile disease severity, we treated mice with three combinations of antibiotics prior to infection. Mice given clindamycin alone became colonized but displayed no tissue pathology while severe disease, exemplified by weight loss and inflammatory tissue damage occurred in animals given a combination of five antibiotics and clindamycin. Animals given only the five antibiotic cocktails showed only transient colonization and no disease. C. difficile colonization was associated with a reduction in bacterial diversity, an inability to amplify bile salt hydrolase (BSH) sequences from fecal DNA and a relative increase in primary bile acids (pBA) in cecal lavages from infected mice. Further, the link between BSA modification and the microbiome was confirmed by the isolation of strains of Lactobacillus murinus that modified primary bile acids in vitro, thus preventing C. difficile germination. Interestingly, BSH activity did not correlate with disease severity which appeared linked to alternations in mucin, which may indirectly lead to increased exposure of the epithelial surface to inflammatory signals. These data confirm the role of microbial metabolic activity in protection of the gut and highlights the need for greater understanding the function of bacterial communities in disease prevention.

Keywords:

• Bile salt metabolism
• Clostridium difficile
• germination
• antibiotics
• disease severity

Acknowledgments

CJ was funded as part of a BBSRC DTP program. AB was funded by the Wellcome Trust (086418). JRM receives financial support from the National Institute for Health Research (NIHR) Imperial Biomedical Research Centre (BRC) based at Imperial College Healthcare NHS Trust and Imperial College London. UZI is funded by a NERC independent Research Fellowship (NE/L011956/1). JVL is funded by MRC New Investigator (MR/P002536/1) and ERC Starting Grant (715662).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Contributions

The work was performed by CJ as part of the requirements for a PhD qualification from the University of Glasgow, UK. CJ performed the experiments with support from AB, JS, JI, and DC; JVL analyzed the composition of bile salts from intestinal samples; UZI provided bioinformatic support for microbiome characterization; CJ, JRM and GRD interpreted the results and wrote the manuscript. All authors contributed to the editing of the manuscript.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.

Additional information

Funding

This work was supported by the Biotechnology and Biological Sciences Research Council [DTP programme BB/J013854/1]; National Institute for Health Research [Imperial Biomedical Research Centre]; Natural Environment Research Council [NE/LO11956/1]; Wellcome Trust [086418].